Functional interaction between bovine rhodopsin and G protein transducin

To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.     

Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other G(o)-coupled receptors belonging to the rhodopsin superfamily showed significant G(o) activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other G(o)-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu(134), Val(138), and Cys(140)) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.     

Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.     

The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.     

Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.     

Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.     

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.     

Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.     

Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.     

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.     

Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin

Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

A型γ-氨基丁酸受体α1亚基Cys~(166)-Leu~(296)片段的配体结合和结构性质

研究A型γ 氨基丁酸受体 (γ aminobutyricacidtypeA ,GABAAreceptor)α1亚基Cys166 Leu2 96片段的苯并二氮杂 (benzodiazepine ,BZ)结合位点及其结构特性 ,了解该片段结构与功能的关系 .利用PfuDNA多聚酶依赖的点突变技术将该片段的每一残基用丙氨酸替代 ,通过E .coli体系过表达 ,纯化得到各种突变蛋白 .运用圆二色性 (circulardichroism ,CD)技术测定突变蛋白的二级结构 ,借助荧光各向异性 (fluorescenceanisotropy ,FA)、荧光共振能量转移 (fluorescenceresonanceenergytrans fer,FRET)技术测定其与BZ荧光配基Bodipy FLRo 1986 (BFR)的结合强弱 .通过与野生型的比较 ,确定其残基是否与结构和或结合相关 .结果显示 ,突变体R191A、G2 12A、S2 13A、R2 14A及V2 79A的结合能力减弱 2～ 3倍 ,除V2 79A显著增加α螺旋外均无二级结构的改变 .E193A、S2 78A、V2 79A和P2 80A的α螺旋显著增多 ,N2 75A和R2 76A的α螺旋则显著减少 .推测Cys166 Leu2 96的Arg191,Gly2 12 ,Ser2 13 和Arg2 14 可能位于BZ的结合袋 ,其第 4个环区 (Glu2 10 Asn2 16)与结合密切相关 .Glu193 、Ser2 78和Pro2 80 参与维持β折叠结构 ,而Asn2 75和Arg2 76参与维持α螺旋结构 .Cys166 Leu2 96的第 9个环区 (Asn2 75 Pro2 80 )对其结     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study.

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.     

The Role of Arrestin α-Helix I in Receptor Binding

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors, thereby preventing G protein coupling and often switching signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action has not been determined yet. Here we show that several mutations in the helix itself and in adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to light-activated phosphorylated rhodopsin (P-Rh^?). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh^? and light-activated unphosphorylated rhodopsin, these mutations reduce the stability of the arrestin complex with P-Rh^?, but not with light-activated unphosphorylated rhodopsin. Using site-directed spin labeling, we found that the local structure around α-helix I changes upon binding to rhodopsin. However, the intramolecular distances between α-helix I and adjacent β-strand I (or the rest of the N-domain), measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping β-strand I, which carries several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.     

Residues in the first extracellular loop of a G protein-coupled receptor play a role in signal transduction

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were used as a model system to study ligand-receptor interaction. Cys-scanning mutagenesis on each residue of EL1, the first extracellular loop of Ste2p, was used to generate a library of 36 mutants with a single Cys residue substitution. Mutation of most residues of EL1 had only negligible effects on ligand affinity and biological activity of the mutant receptors. However, five mutants were identified that were either partially (L102C and T114C) or severely (N105C, S108C, and Y111C) compromised in signaling but retained binding affinities similar to those of wild-type receptor. Three-dimensional modeling, secondary structure predictions, and subsequent circular dichroism studies on a synthetic peptide with amino acid sequence corresponding to EL1 suggested the presence of a helix corresponding to EL1 residues 106 to 114 followed by two short beta-strands (residues 126 to 135). The distinctive periodicity of the five residues with a signal-deficient phenotype combined with biophysical studies suggested a functional involvement in receptor activation of a face on a 3(10) helix in this region of EL1. These studies indicate that EL1 plays an important role in the conformational switch that activates the Ste2p receptor to initiate the mating pheromone signal transduction pathway.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding

Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in the absence or presence of ligand. In right-side-out membrane vesicles, Cys residues in the cytoplasmic halves of both helices react with NEM in the absence of ligand, while Cys residues in the periplasmic halves do not. Five Cys replacement mutants at the periplasmic end of helix V and one at the cytoplasmic end of helix V label only in the presence of ligand. Interestingly, in addition to native Cys148, a known binding-site residue, labeling of mutant Ala122 --> Cys, which is located in helix IV across from Cys148, is markedly attenuated by ligand. Furthermore, alkylation of the Ala122 --> Cys mutant blocks transport, and protection is afforded by substrate, indicating that Ala122 is also a component of the sugar binding site. Methanethiosulfonate ethylsulfonate, an impermeant thiol reagent shown clearly in this paper to be impermeant in E. coli spheroplasts, was used to identify substituted Cys side chains exposed to water and accessible from the periplasmic side. Most of the Cys mutants in the cytoplasmic halves of helices IV and V, as well as two residues in the intervening loop, are accessible to the aqueous phase from the periplasmic face of the membrane. The findings indicate that the cytoplasmic halves of helices IV and V are more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that exhibit ligand-induced changes are located for the most part in the vicinity of the residues directly involved in substrate binding, as well as the cytoplasmic loop between helices IV and V.     

Congenital hypogonadotropic hypogonadism due to GnRH receptor mutations in three brothers reveal sites affecting conformation and coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gα(q/11) signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.